Systematic Synthesis of Dinucleotides and Trinucleotides with RNases U2 N1 and a Non-Specific RNase from B. subtilis

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Abstract
Using reverse transesterification by three RNases with different base specificities, that is RNases U2 N1 and B. subtilis RNase, a method for the systematic synthesis for all 16 possible dimers and 50 trimers out of 64 in all was established. The yield of dimer was dependent on the ratio of phosphate acceptor to donor (A/D) and the total substrate concentration, in addition to various other conditions. A/D and the acceptor concentration were preferably more than 10 and 0.25 M, respectively. Therefore, the yield of dimer was dependent on the solubility of the acceptor, when it was less than 0.25 M. Trinucleotide were also synthesized with RNases U2 or N1 as follows; I. X−Y>p+N or II. X>p+Y−N. The mode and yield of trimer synthesis were quite similar to those of the corresponding dimer synthesis with the same joining site sequence. B. subtilis RNase was not useful for trimer synthesis, because the rate of degradation of dinucleotide used as the acceptor or donor was faster than that of reverse transesterification. The yield of dimer synthesis was 50–70% using a pyrimidine derivative as an acceptor and 10–30% using a purine derivative. The yields of trimer in type I reaction were 60–80% in cases with a pyrimidine derivative and 10–30% in cases with a purine derivative, while those in type II reaction were about 20% with pyrimidine as the Y base in acceptor Y-N, and about 10% with purine.